This invention relates to the recombinant synthesis and purification of protein antibodies that influence survival, proliferation, differentiation or maturation of hematopoietic cells, especially platelet progenitor cells and to antibodies that influence the growth and differentiation of cells expressing a protein kinase receptor. This invention also relates to the cloning and expression of nucleic acids encoding antibody ligands (thrombopoietin receptor agonist antibodies) capable of binding to and activating a thrombopoietin receptor such as c-mpl, a member of the cytokine receptor superfamily. This invention further relates to the use of these antibodies alone or in combination with other cytokines to treat immune or hematopoietic disorders including thrombocytopenia and to uses in assays.
In 1994 several groups reported the isolation and cloning of thrombopoietin (F. de Sauvage et al., Nature 369:533 (1994); S. Lok et al., Nature 369:565 (1994); T D. Bartley et al., Cell 77:1117 (1994); Y. Sohma et al., FEBS Letters 353:57 (1994); D J. Kuter et al., Proc. Natl. Acad. Sci. 91:11104 (1994)). This was the culmination of more than 30 years of research initiated in the late 50""s when Yamamoto (S. Yamamoto, Acta Haematol Jpn. 20:163-178. (1957)) and Kelemen (E. Kelemen et al., Acta Haematol (Basel). 20:350-355 (1958)) proposed that physiological platelet production is controlled by a humoral factor termed xe2x80x9cthrombopoietinxe2x80x9d(TPO). Although routinely detected in urine, plasma and serum from thrombocytopenic animals and patients, as well as kidney cell conditioned media, purification of TPO proved to be a daunting task (for a review see M S. Gordon et al., Blood 80:302 (1992); W. Vainchenker et al., Critical Rev. Oncology/Hematology 20:165 (1995)). In the absence of purified TPO and the apparent fact that numerous plieotrophic cytokines affected megakaryocytopoiesis (M S. Gordon et al., Blood 80:302 (1992); W. Vainchenker et al., Critical Rev. Oncology/Hematology 20:165 (1995)), the existence of a lineage specific factor that regulated platelet production was doubted until the discovery of the orphan cytokine receptor c-Mpl in 1990 (M. Souyri et al., Cell 63:1137 (1990); I. Vigon et al., Proc. Natl. Acad. Sci. 89:5640 (1992)). The expression of c-Mpl was found to be restricted to progenitor cells, megakaryocytes and platelets, and c-Mpl antisense oligonucleotides selectively inhibited in vitro megakaryocytopoiesis (M. Methia et al., Blood 82:1395 (1993)). From this it was postulated that c-Mpl played a critical role in regulating megakaryocytopoiesis and that its putative ligand may be the long sought TPO (M. Methia et al., supra). Following this discovery several groups utilizing c-Mpl ligand specific cell proliferation assays and c-Mpl as a purification tool isolated and cloned the ligand for c-Mpl (F. de Sauvage a al., supra; S. Lok et al., supra; T D. Bartley et al., supra). In addition two other groups independently reported the purification of the Mpl-ligand using standard chromatography techniques and megakaryocyte assays (Y. Sohma et al., supra, D J. Kuteret al., supra). In the years since its reported discovery numerous studies clearly indicate that the Mpl-ligand possess all the characteristics that have long been attributed to the purported regulator of megakaryocytopoiesis and thrombopoiesis and consequently, is now referred to as TPO. The Mpl ligand is currently referred to as either TPO or as megakaryocyte growth and differentiation factor (MGDF).
Human TPO consists of 332 amino acids that can be divided into 2 domains; an amino terminal domain of 153 amino acids showing 23% identity (50% similarity) to erythropoietin (EPO) and a unique 181 amino acid C-terminal domain that is highly glycosylated ((F. de Sauvage et al., supra; S. Lok et al., supra; T D. Bartley et al., supra). The EPO-like domain of TPO contains 4 cysteines, 3 of which are conserved with EPO. The first and last and the two middle cysteines form two disulfide bridges, respectively, which are both required for activity (T. Kato et al., Blood 86 (suppl 1):365 (1995)). None of the Asn-linked glycosylation sites present in EPO are conserved in the EPO-like domain of TPO, however, the EPO-like domain of recombinant TPO (rTPO) contains 2-3 O-linked glycosylations (M. Eng et al., Protein Science 5(suppl 1):105 (1996)). A recombinant truncated form of TPO (rTPO153), consisting of only the EPO-like domain, is fully functional in vitro, indicating that this domain contains all the required structural elements to bind and activate Mpl (F. de Sauvage et al., supra; D L. Eaton et al., Blood 84(suppl 1):241 (1994)). The carboxy terminal domain of TPO contains 6 N-linked and 18 O-linked glycosylated sites and is rich in proline, serine and threonine (M. Eng et al., supra). The function of this domain remains to be elucidated. However, because of its high degree of glycosylation this region may act to stabilize and increase the half life of circulating TPO. This is supported by the observation that rTPO153 has a half life of 1.5 hours compared to 18-24 hours for full length glycosylated rTPO (G R. Thomas et al., Stem Cells 14(suppl 1) (1996).
The two domains of TPO are separated by a potential dibasic proteolytic cleavage site that is conserved among the various species examined. Processing at this site could be responsible for releasing the C-terminal region from the EPO domain in vivo. The physiological relevance of this potential cleavage site is unclear at this time. Whether TPO circulates as an intact full length molecule or as a truncated form is also equivocal. When aplastic porcine plasma was subjected to gel filtration chromatography, TPO activity present in this plasma resolved with a Mr. of xcx9c150,000 ((F. de Sauvage et al., supra). Purified full length rTPO also resolves at this Mr., whereas the truncated forms resolve with Mr. ranging from 18,000-30,000. Using TPO ELISAs that selectively detect either full length or truncated TPO it has also been shown that full length TPO is the predominant form in the plasma of marrow transplant patients (Y G. Meng et al., Blood 86(suppl. 1):313 (1995)).
Prior to the discovery of c-Mpl and the isolation of TPO, it was thought that megakaryocytopoiesis was regulated at multiple cellular levels (M S. Gordon et al., supra; W. Vainchenker et al., supra; Y G. Meng et al., supra). This hypothesis was based on the observation that certain hematopoietic growth factors stimulated proliferation of megakaryocyte progenitors while others primarily affected maturation (M S. Gordon et al., supra; W. Vainchenker et al., supra; Y G. Meng et al., supra). Other data indicated that plasma from thrombocytopenic animals contained distinct activities that either affected proliferation (meg-CSF) or maturation (TPO) of megakaryocytes (R J. Hill et al., Exp. Hematol 20:354 (1992)). Wendling and her colleagues (F. Wendling et al., Nature 369:571 (1994)) initially dispelled this theory by demonstrating that all the megakaryocyte colony-stimulating and thrombopoietic activities in thrombocytopenic plasma could be neutralized by soluble Mpl. This indicated that these activities are due to a single factor, the Mpl-ligand. Numerous studies have now shown that recombinant forms of TPO not only induce proliferation of progenitor megakaryocytes but also their maturation (K. Kaushansky et al., Nature 369:568 (1994); F C. Zeigler et al., Blood 94:4045 (1994); V C. Broudy et al., Blood 85:1719 (1995); J L. Nichol et al., J. Clin. Invest. 95:2973 (1995); N. Banu et al., Blood 86:1331 (1995); N. Debili et al., Blood 86:2516 (1995); P. Angchaisuksiri et al., Br. J. Haematol. 93:13 (1996); E S. Choi et al., Blood 85:402 (1995)). Human CD34+, CD34+,CD41+ cells (F C. Zeigler et al., supra; V C. Broudy et al., supra; J L. Nichol et al., supra; N. Banu et al., supra;) or purified murine stem cells (sca+,linxe2x88x92, kit+) (K. Kaushansky et al., supra: F C. Zeigler et al., supra) cultured with rTPO selectively differentiate to megakaryocytes. rTPO induces the differentiation and proliferation of megakaryocyte colonies in semisolid cultures and single megakaryocytes in liquid suspension cultures. This activity appears to be a direct effect of TPO as limiting dilution experiments show a direct correlation between progenitors seeded and megakaryocytes obtained (N. Debili et al., supra). In addition comparable results are obtained in serum free or serum containing culture conditions (N. Banu et al., supra; N. Debili et al., supra; P. Angchaisuksiri et al., supra;). These observations indicate that neither accessory cells or serum components are required for TPO to induce megakaryocyte growth and differentiation in vitro.
The effect of rTPO on the megakaryocyte maturation process is dramatic. rTPO induces highly purified murine or human progenitor cells in liquid culture to differentiate into very large mature polyploid megakaryocytes (F C. Zeigler et al., supra; V C. Broudy et al., supra; J L. Nichol et al., supra; N. Debili et al., supra). Megakaryocytes from such cultures exhibit ploidy of 4N-16N with ploidy classes of 64N and 128N also being detected in these cultures (N. Debili et al., supra). In addition, megakaryocytes produced from these cultures undergo a terminal maturation process and appear to develop proplatelets and shed platelet like structures into the medium (F C. Zeigler et al., supra; N. Debili et al., supra; E S. Choi et al., supra). Significantly, the platelets produced from such cultures have been shown to be morphologically and functionally indistinct from plasma-derived platelets (E S. Choi et al., supra).
Although, rTPO appears to act directly on hematopoietic progenitors to induce megakaryocyte differentiation, it also acts synergistically and additively with early and late acting hematopoietic factors. In murine megakaryocytopoiesis assays IL-11, kit ligand (KL) or EPO act synergistically and IL-3 and IL-6 act additively with rTPO to stimulate proliferation of megakaryocyte progenitors (V C. Broudy et al., supra). In human megakaryocytopoiesis assays IL-3 and IL-6 effects are additive to rTPO, while KL acts synergistically with rTPO (J L. Nichol et al., supra; N. Banu et al., supra; N. Debili et al., supra; P. Angchaisuksiri et al., supra). None of the cytokines mentioned above affect the megakaryocyte maturational activity of rTPO.
The initial studies with rTPO clearly indicate that TPO predominantly affects the megakaryocytic lineage. However, like all other hematopoietic regulators, TPO affects other hematopoietic lineages as well. In the presence of EPO, rTPO has been shown to enhance erythroid burst (BFU-E) formation in human CD34+ colony assays (M. Kobayashi et al., Blood 86:2494 (1995); T. Papayannopoulou et al., Blood 87:1833 (1996)). The burst promoting activity of rTPO is comparable to GM-CSF and KL and increases both the number and size of BFU-E colonies (M. Kobayashi et al., supra). In addition rTPO also stimulates CFU-E development, indicating that TPO acts on both early and late erythroid progenitors (M. Kobayashi et al., supra; T. Papayannopoulou et al., supra). In the absence of EPO, however, rTPO has no effect on etythropoiesis. An effect of rTPO on myeloid colony growth in normal hematopoietic cultures has not been demonstrated in vitro, however.
rTPO has a dramatic effect on platelet production when administered to normal animals. Pharmacological doses of recombinant forms of TPO cause as much as a 10 fold increase in platelet levels in mice and non-human primates (E F. Winton et al., Exp. Hematol. 23:879 (1995); A M. Farese et al., Blood 86:54 (1995); K H. Sprugel et al., Blood 86(suppl 1):20 (1995); L A. Harker et al., Blood 87:1833 (1996); K. Kaushansky et al., Exp. Hematol. 24:265 (1996); T R. Ulich et al., Blood 87:5006 (1996); K. Ault et al., Blood 86(suppl 1): 367 (1995); N C. Daw et al., Blood 86 (suppl 1):5006 (1995)). This effect of rTPO is due to an increase in the synthesis of new platelets as reticulated platelets increase within 24 hours after rTPO administration (K. Ault et al., supra). Preceding this effect is a dramatic increase in CFU-MK in both the marrow and spleen (A M. Farese et al., supra; K. Kaushansky et al., supra; T R. Ulich et al., supra). Megakaryocytes from rTPO treated animals exhibit a higher mean ploidy and are larger in size than megakaryocytes from control animals. These later two observations again demonstrate the proliferative and maturational activities of TPO on the megakaryocytic lineage. Because the effect of TPO on megakaryocytes precedes its effect on platelet production it has been suggested that TPO primarily affects megakaryocyte progenitors rather than inducing platelet release from mature megakaryocytes (N C. Daw et al., supra). No significant effect on red blood cell (RBC) or white blood cell (WBC) production occurs in normal animals following rTPO administration. However, rTPO treatment caused an expansion of BFU-E and CFU-GM and a redistribution CFU-E in normal mice (K. Kaushansky et al., supra) and expanded CFU-mixed in rhesus monkeys (A M. Farese et al., supra).
Even though rTPO dramatically stimulates platelet production, it only has a modest effect on platelet function. In vitro studies show that rTPO has no effect on platelet aggregation itself, but does enhance agonist induced aggregation (G. Montrucchio et al., Blood 87:2762 (1996); A. Oda et al., Blood 87:4664 (1996); C F. Toombs et al., Thromb. Res. 80:23 (1995); C F. Toombs et al., Blood 86(suppl 1):369 (1995)). rTPO appears to sensitize platelets making them moderately more responsive to aggregation agonist. This raises the possibility that rTPO may have prothrombotic effects in vivo. However, an increase in thrombotic episodes in animals treated with rTPO has never been observed, even when platelet levels were 4-10 fold above normal. In vivo thrombosis models also indicate that elevated platelet levels following rTPO treatment is not associated with an increase in platelet dependent thrombosis (L A. Harker et al., supra, C F. Toombs et al., supra). These results indicate that stimulation of platelet production by rTPO will unlikely be associated with an increase in thrombo-occulsive events.
The involvement of c-Mpl and TPO in the control of platelet production and its effect on other hematopoietic lineages is further demonstrated by the phenotype of mice deficient in either the c-mpl or the TPO genes (W S. Alexander et al., Blood 87:2162 (1996); F J. de Sauvage et al., J. Exp. Med. 183:651 (1996); A L. Gurney et al., Science 265:1445 (1994)). In both cases a dramatic 85 to 90% drop in platelet counts is observed with a similar decrease of megakaryocytes in the spleen and bone marrow. In addition, the megakaryocytes of the knockout mice are smaller and exhibit a lower ploidy than those of control mice. The similarity in phenotype observed for these knock-outs (KO) indicates that the system is non-redundant and that there is probably only one receptor for TPO and one ligand for c-Mpl. Although the platelet number is reduced in the KO mice their platelets appear normal, both structurally and functionally, and are sufficient to prevent overt bleeding. The genes and factors involved in the production of this basal level of platelets and megakaryocytes still remain to be identified. However, treatment of either the TPO or c-mpl knockout mice with other cytokines with megakaryopoietic activity (IL-6, IL-11 and stem cell factor) results in a modest stimulation of platelet production (A L. Gurney et al., supra). This suggest that these cytokines do not require TPO or c-mpl to exert their thrombopoietic activity and, therefore, may be involved in the maintenance of a basal level of megakaryocytes and platelets.
Comparison of CFU-megakaryocyte (CFU-Meg) from TPO or c-mpl deficient and normal mice shows that the number of megakaryocytes progenitors is decreased in both knock-outs compared to control, suggesting that TPO acts on very early megakaryocyte progenitors. In addition, both erythroid and myeloid progenitors are also reduced in the TPO and c-Mpl knockout mice (W S. Alexander et al., supra, K. Carver-Moore et al., 88:803 (1996)). This reduction in progenitors from all lineages indicates that TPO probably acts on a very early pluripotent progenitor cell. The involvement of TPO and c-Mpl at an early stage of hematopoiesis correlates with the detection of c-Mpl expression in AA4+Sca+ murine stem cell population (F C. Zeigler et al., supra). The effect of TPO on this most primitive stem cell population still remains to be investigated, however, preliminary data indicate that TPO may directly affect the proliferation of primitive murine hematopoietic stem or progenitor cells (E. Stinicka et al., Blood 87:4998 (1996); M. Kobayashi et al., Blood 88:429 (1996); H. Ku et al., Blood 87:4544 (1996)). This, in part, may explain the effect TPO has on erythropoiesis and myelopoiesis in vitro and in vivo.
It has long been observed that an inverse correlation exists between plasma megakaryopoietic and thrombopoietic activity and platelet levels (reviewed in T P. McDonald, Am. J. Pediatr. Hematol./Oncol. 14:8 (1992)). TPO specific ELISAs and cell proliferation assays have now confirmed that TPO levels increase and decrease inversely with platelet mass (J L. Nichol et al., supra, E V B. Emmons et al., Blood 87:4068 (1996); H. Oh et al., Blood 87:4918 (1996); M. Chang et al., Blood 86(suppl 1):368 (1995)). Unlike erythropoietin, however, TPO does not appear to be regulated at the transcriptional level, but rather by platelet mass. This was initially proposed de Gabriele and Pennington (G. de Gabriele et al., Br. J. Haematol. 13:202 (1967); G. de Gabriele et al., Br. J. Haematol. 13:210 (1967)) and subsequently confirmed by Kuter and Rosenberg (D J. Kuter et al., Blood 84:1464 (1994)) who showed direct regulation of circulating TPO levels by exogenously administering platelets to thrombocytopenic mice. More recently, it was demonstrated that TPO mRNA levels in thrombocytopenic mice are not increased even though TPO levels are elevated by at least 10 fold (P J. Fielder et al., Blood 87:2154 (1996); R. Stoffel et al., Blood 87:567 (1996)). In addition, the gene dosage effect observed in TPO heterozygous knockout mice refute the regulation of TPO production by platelet mass (F J. de Sauvage et al., supra). Taken together, these results strongly support the hypothesis that TPO expression is constitutive and it is the sequestering by platelets that regulates TPO levels. Platelets bind TPO with high affinity (Kd(100-400 pM) and internalize and degrade TPO (P J. Fielder et al., supra). Platelets from c-Mpl knockout mice do not bind TPO and the clearance of TPO by these mice is S fold slower than that observed for wild type mice (P J. Fielder et al., supra). These results indicate that TPO clearance is mediated by platelet binding via c-Mpl. It is also likely that megakaryocyte mass plays a role in regulating circulating TPO levels. This is supported by the observation that both ITP patients and mice deficient in the NF-E2 transcription factor are highly thrombocytopenic, exhibit megakaryocytosis, but have normal TPO levels (E V B. Emmons et al., supra; R A. Shivdasani et al., Cell 81:695 (1995)). In situ studies with radiolabeled TPO show that marrow megakaryocytes of the NF-E2 mice bind significant amounts of labeled TPO (R A. Shivdasani et al., Blood submitted (1996)). The phenotype of the ITP and NF-E2 knockout mice, therefore, suggest that binding of TPO to megakaryocytes may also regulate TPO levels.
The dramatic effect of rTPO on platelet production in normal mice and monkeys and subsequent clinical trials indicate that rTPO is clinically useful in alleviating thrombocytopenia associated with myelosuppressive and mycloablative therapies for cancer patients. In several myelosuppressive and mycloablative murine and monkey preclinical models recombinant forms of TPO have been shown to significantly affect platelet recovery. In mice treated with carboplatin and sublethal irradiation in combination (J P Leonard et al., Blood 83:1499 (1994)), daily treatment with rTPO both reduced the severity of the platelet nadir and accelerated platelet recovery by 10-12 days when compared to excipient treated animals (G R. Thomas et al., supra; K. Kaushansky et al., supra, M M. Hokom et al., Blood 86:4486 (1995)). Similar results were obtained in a murine sublethal irradiation model (G R. Thomas et al., supra). In murine mycloablative transplantation models rTPO has been shown to reduce the extent of the nadir and accelerate platelet recovery by 2-3 weeks (G R. Thomas et al., supra; K. Kabaya et al., Blood 86(suppl l):114 (1995); G. Molineux et al., Blood 86(suppl 1):227 (1995)). Treatment of sublethally irradiated rhesus monkeys with rTPO accelerated platelet recovery by 3 weeks and prevented platelet nadirs below 40,000 (A M. Farese et al., J. Clin. Invest. 97:2145 (1996); K J. Neelis et al., Blood 86(suppl 1):256 (1995)). Even more impressively, rTPO completely prevented post-chemotherapy thrombocytopenia following the treatment of rhesus monkeys with hepsulfam (A M. Farese et al., supra). In contrast to these promising results, two groups have reported that rTPO had no effect on the hematopoietic recovery of lethally irradiated mice or monkeys rescued with a marrow transplant (K J. Neelis et al, supra; W E. Fibbe et al., Blood 86:3308 (1995)). The reason for this discrepancy is unclear, however it is possible that lethal radiation may destroy stromal cells or components essential for TPO activity in vivo. In support of this, lethally irradiated mice transplanted with marrow cells from rTPO treated donor mice show accelerated recovery of platelets and RBCs, however, post-transplant administration of rTPO had no further effect on this accelerated recovery (W E. Fibbe et al., supra). This result suggests that although the transplanted cell population was enriched for megakaryocyte progenitors, TPO had no effect on these progenitors in a lethally irradiated marrow.
Although rTPO only modestly affects erythroid and myeloid lineages in normal mice it dramatically accelerates the recovery of all progenitor classes in myelosuppressed mice and monkeys resulting in a significant acceleration of RBC and WBC recovery (K. Kaushansky et al., supra; A M. Farese et al., supra; K. Kaushansky et al., J. Clin. Invest. 96:1683 (1995)). The effect of rTPO on neutrophil recovery has been shown to be additive to that of G-CSF (A M. Farese et al., supra). These results indicate that the clinical utility of rTPO may be broader than originally anticipated.
The difference between the effect of rTPO on hematopoiesis in normal and myelosuppressed animals is likely due to the change in the cytokine environment that occurs following myelosuppressive therapy. It is likely that elevated levels of EPO, G-CSF or other cytokines essential for erythropoiesis and myelopoiesis present following myelosuppressive treatment interact with rTPO to have a multilineage effect (K. Kaushansky et al., supra). In normal mice the level of these cytokines are insufficient and the effects of rTPO on erythroid and myeloid lineages are less significant. This hypothesis is supported by the above mentioned synergistic interaction of rTPO and EPO to stimulate in vitro erythropoiesis (E S. Choi et al., supra). It has also been proposed that production of hemopoietic factors from megakaryocytes themselves may also play a role in the multilineage effect of rTPO (A M. Farese et al., supra).
In the above mentioned animal studies rTPO was administered daily for 14-28 days, which was based on previous experience in dosing other hematopoietic growth factors. However, it has recently been shown that a single dose of rTPO following myelosuppressive treatment of mice with carboplatin and sublethal irradiation is as effective as multiple doses in reducing nadirs and accelerating platelet and RBC recovery (G R. Thomas et al., supra). This effect is likely due to the potency and long half life of rTPO.
(G R. Thomas et al., supra). This is supported by the fact that single doses of unglycosylated rTPO153 are not effective in this model. These observations indicate that the frequency of rTPO dosing required to affect hematopoietic recovery following myelosuppressive treatment may be significantly less than that for other currently used cytokines.
Early results from human clinical trails show that rTPO also stimulates platelet production in humans. In phase I trials, a pegylated and truncated form of rTPO (MGDF) administered daily for 10 days at 0.03-5.0 xcexcg/kg to cancer patients prior to chemotherapy caused up to a four fold increase in circulating platelet levels (R. Basser et al., Blood 86(suppl 1): 257 (1995); J E J. Rasko et al., Blood 86(suppl 1):497 (1995)).Similarly, patients given a single dose of rTPO had platelet levels increase by four fold (S. Vaden-Raj et al., Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in cancer patients. Submitted. (1996)). In both studies platelet increases are observed by day four and peak about 12-16 days later. No drug related toxicity""s were reported and, although platelet levels greater then 1xc3x97106/xcexcl were observed in some of the patients, no thrombotic events were observed. This indicates that TPO will be well tolerated in humans. In myelosuppressed patients, pegylated rTPO153(MGDF) given post chemotherapy has been shown to reduce the extent of the platelet nadir following chemotherapy (G. Begley et al., Proceedings of ASCO 15:271 (1996); M. Fanucchi et al., Proceedings of ASCO 15:271 (1996)). As seen in the preclinical animals studies, TPO also expanded marrow progenitors of megakaryocyte, erythroid, myeloid and multipotential lineages (S. Vaden-Raj et al., supra). This later observation suggests that rTPO may be useful as a priming agent.
It is believed that the proliferation and maturation of hematopoietic cells is tightly regulated by factors that positively or negatively modulate pluripotential stem cell proliferation and multilineage differentiation. These effects are mediated through the high-affinity binding of extracellular protein factors (ligands) to specific cell surface receptors. These cell surface receptors share considerable homology and are generally classified as members of the cytokine receptor superfamily. Members of the superfamily include receptors for: IL-2 (b and g chains) (Hatakeyama et al., Science, 244:551-556 (1989); Takeshita et al., Science, 257:379-382 (1991)), IL-3 (Itoh et al., Science, 247:324-328 (1990); Gorman et al., Proc. Natl. Acad. Sci. USA, 87:5459-5463 (1990); Kitamura et al., Cell, 66:1165-1174 (1991a); Kitamura et al., Proc. Natl. Acad. Sci. USA, 88:5082-5086 (1991b)), IL-4 (Mosley et al., Cell, 59:335-348 (1989), IL-5 (Takaki et al., EMBO J., 9:4367-4374 (1990); Tavernier et al., Cell, 66:1175-1184 (1991)), IL-6 (Yamasaki et al., Science, 241:825-828 (1988); Hibi et al., Cell, 63:1149-1157 (1990)), IL-7 (Goodwin et al., Cell, 60:941-951 (1990)), IL-9 (Renault et al., Proc. Natl. Acad. Sci. USA, 89:5690-5694 (1992)), granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gearing et al., EMBO J., 8:3667-3676 (1991); Hayashida et al., Proc. Natl. Acad. Sci. USA, 244:9655-9659 (1990)), granulocyte colony-stimulating factor (G-CSF) (Fukunaga et al. Cell, 61:341-350 (1990a); Fukunaga et al., Proc. Natl. Acad. Sci. USA, 87:8702-8706 (1990b); Larsen et al., J. Exp. Med, 172:1559-1570 (1990)), EPO (D""Andrea et al., Cell, 57:277-285 (1989); Jones et al., Blood, 76:31-35 (1990)), Leukemia inhibitory factor (LIF) (Gearing et al., EMBO J., 10:2839-2848 (1991)), oncostatin M (OSM) (Rose et al., Proc. Natl. Acad. Sci. USA, 88:8641-8645 (1991)) and also receptors for prolactin (Boutin et al., Proc. Natl. Acad. Sci. USA, 88:7744-7748 (1988); Edery et al., Proc. Natl. Acad. Sci. USA, 86:2112-2116 (1989)), growth hormone (GH) (Leung et al., Nature, 330:537-543 (1987)) and ciliary neurotrophic factor (CNTF) (Davis et al., Science, 253:59-63 (1991).
Members of the cytokine receptor superfamily may be grouped into three functional categories (for review see Nicola et al., Cell, 67:1-4 (1991)). The first class comprises single chain receptor such as erythropoietin receptor (EPO-R) or granulocyte colony stimulating factor receptor (G-CSF-R), which bind ligand with high affinity via the extracellular domain and also generate an intracellular signal. A second class of receptors, so called a-subunits, includes interleukin-6 receptor (IL6-R), granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R), interleukin-3 receptor (IL3-Ra) and other members of the cytokine receptor superfamily. These a-subunits bind ligand with low affinity but cannot transduce an intracellular signal. A high affinity receptor capable of signaling is generated by a heterodimer between an a-subunit and a member of a third class of cytokine receptors, termed b-subunits, e.g., bC, the common b-subunit for the three a-subunits of IL-3-R, IL-5-R and GM-CSF-R (Nicola N. A. et. al. Cell 67:1-4 (1991)).
Evidence that mpl is a member of the cytokine receptor superfamily comes from sequence homology (Gearing, EMBO J., 8:3667-3676 (1988); Bazan, Proc. Natl. Acad. Sci. USA, 87:6834-6938 (1990); Davis et al., Science, 253:59-63 (1991) and Vigon et al., Proc. Natl. Acad. Sci. USA, 89:5640-5644 (1992)) and its ability to transduce proliferative signals.
Deduced protein sequence from molecular cloning of murine c-mpl reveals this protein is homologous to other cytokine receptors. The extracellular domain contains 465 amino acid residues and is composed of two subdomains each with four highly conserved cysteines and a particular motif in the N-terminal subdomain and in the C-terminal subdomain. The ligand-binding extracellular domains are predicted to have similar double b-barrel fold structural geometries. This duplicated extracellular domain is highly homologous to the signal transducing chain common to IL-3, IL-5 and GM-CSF receptors as well as the low-affinity binding domain of LIF (Vigon et al., Oncogene, 8:2607-2615 (1993)). Thus mpl may belong to the low affinity ligand binding class of cytokine receptors.
A comparison of murine mpl and mature human mpl P, reveals these two proteins show 81% sequence identity. More specifically, the N-terminus and C-terminus extracellular subdomains share 75% and 80% sequence identity respectively. The most conserved mpl region is the cytoplasmic domain showing 91% amino acid identity, with a sequence of 37 residues near the transmembrane domain being identical in both species. Accordingly, mpl is reported to be one of the most conserved members of the cytokine receptor superfamily (Vigon supra).
Activation of certain hematopoietic receptors is believed to cause one or more effects including; stimulation of proliferation, stimulation of differentiation, stimulation of growth and inhibition of apoptosis (Libol et al Proc. Natl. Acad. Sci. 248:378 (1993). Activation of hematopoietic receptors upon ligand binding may be due to dimerization of two or more copies of the receptor. In addition to the naturally occurring ligand causing this dimerization, agonist antibodies may also activate receptors by crosslinking or otherwise causing dimerization of a receptor. Such antibodies are useful for the same indications as the natural ligand and may have advantageous properties such as a longer half-life. An example of a monoclonal antibody to a cytokine receptor that activates the erythropoietin receptor (EPO-R) is described in WO 96/03438 (published Feb. 8, 1996). These agonist antibodies to EPO-R are about 3-4 orders of magnitude weaker in activity based on weight than the natural EPO ligand.
There is a current and continuing need to isolate and identify molecules, especially antibodies, fragments and derivatives thereof, capable of stimulating proliferation, differentiation and maturation and/or modulation of apoptosis of cells, for example hematopoietic cells, including megakaryocytes or their predecessors for therapeutic use in the treatment of hematopoietic disorders including thrombocytopenia.
Accordingly, It is an object of this invention to obtain a pharmaceutically or essentially pure antibody or fragments or derivatives thereof capable of stimulating proliferation, differentiation and/or maturation of hematopoietic cells, including megakaryocytes or their predecessors, or to modulate apoptosis of hematopoietic cells.
It is a specific object of the present invention to isolate antibody ligands capable of binding in vivo a hematopoietic growth factor superfamily receptor and to activate the receptor, the antibody having a biological activity equal to or not less than 2 orders of magnitude below that of the naturally occurring ligand on a weight basis.
It is also an object of the present invention to isolate antibody ligands capable of binding to and activating any of the three functional categories of cytokine superfamily receptors (see Nicola et al., Cell, 67:1-4 (1991)).
In one embodiment, the objects of the invention are achieved by providing an antibody or fragment thereof that activates a hematopoietic growth factor superfamily receptor having a biological activity within 2 orders of magnitude (100), preferably within one order of magnitude (10), of the natural ligand on a weight basis. Preferably, the antibody activates the thrombopoietin (TPO) receptor. This antibody, referred to as an agonist antibody, activates a thrombopoietin receptor which preferably comprises a mammalian c-mpl, more preferably human c-mpl. Usually the antibody will be a full length antibody such as an IgG antibody. Suitable presentative fragment agonist antibodies include Fv, ScFv, Fab, F(abxe2x80x2)2 fragments, as well as diabodies and linear antibodies. These fragments may be fused to other sequences including, for example, the Fxe2x80x3 or Fc region of an antibody, a xe2x80x9cleucine zipperxe2x80x9d or other sequences including pegylated sequences or Fc mutants used to improve or modulate half-life. Normally the antibody is a human antibody and may be a non-naturally occurring antibody, including affinity matured antibodies. Representative antibodies that activate c-mpl are selected from the group 12E10, 12B5, 1OF6 and 12D5, and affinity matured derivatives thereof. Other preferred agonist antibodies to c-mpl are selected from the group consisting of Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6, wherein each Ab1-Ab6 contains a VH and VL chain and each VH and VL chain contains complementarity determining region (CDR) amino acid sequences designated CDR1, CDR2 and CDR3 separated by framework amino acid sequences, the amino acid sequence of each CDR in each VH and VL chain of Ab1-Ab6 is shown in Table 1.
Other preferred c-mpl agonist antibodies of this invention include those that activate platelets in a manner similar to TPO or in a manner similar to ADP, collagen and the like. Optionally the c-mpl agonist antibodies of this invention do not activate platelets. The c-mpl agonist antibodies of this invention are used in a w manner similar to TPO.
In another embodiment, substantially pure single chain antibodies are provided which bind to and act as agonist or antagonist antibodies to a cytokine receptor or to a kinase receptor.
The invention also provides a method of obtaining these antibodies, in particular a method of screening a library of phage displayed antibodies, preferably human single chain antibodies.